Differential Roles of Macrophages in Diverse Phases of Skin Repair Tina Lucas, Ari Waisman, Rajeev Ranjan, Jürgen Roes, Thomas Krieg, Werner Müller, Axel Roers and Sabine A. This information is current as Eming of September 24, 2021. J Immunol 2010; 184:3964-3977; Prepublished online 22 February 2010; doi: 10.4049/jimmunol.0903356 http://www.jimmunol.org/content/184/7/3964 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/02/22/jimmunol.090335 Material 6.DC1 http://www.jimmunol.org/ References This article cites 46 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/184/7/3964.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Differential Roles of Macrophages in Diverse Phases of Skin Repair Tina Lucas,* Ari Waisman,† Rajeev Ranjan,* Ju¨rgen Roes,‡ Thomas Krieg,*,x Werner Mu¨ller,{ Axel Roers,‖ and Sabine A. Eming* Influx of macrophages plays a crucial role in tissue repair. However, the precise function of macrophages during the healing response has remained a subject of debate due to their functional dichotomy as effectors of both tissue injury and repair. We tested the hypothesis that macrophages recruited during the diverse phases of skin repair after mechanical injury exert specific functions to restore tissue integrity. For this purpose, we developed a mouse model that allows conditional depletion of macrophages during the sequential stages of the repair response. Depletion of macrophages restricted to the early stage of the repair response (inflam- matory phase) significantly reduced the formation of vascularized granulation tissue, impaired epithelialization, and resulted in minimized scar formation. In contrast, depletion of macrophages restricted to the consecutive mid-stage of the repair response Downloaded from (phase of tissue formation) resulted in severe hemorrhage in the wound tissue. Under these conditions, transition into the subsequent phase of tissue maturation and wound closure did not occur. Finally, macrophage depletion restricted to the late stage of repair (phase of tissue maturation) did not significantly impact the outcome of the repair response. These results demonstrate that macro- phages exert distinct functions during the diverse phases of skin repair, which are crucial to control the natural sequence of repair events. The Journal of Immunology, 2010, 184: 3964–3977. http://www.jimmunol.org/ estoration of skin integrity and homeostasis following blasts, which differentiate into myofibroblasts, and the accumu- injury is a vital process. Across different species, this lation of additional macrophages. Deposition of provisional R event requires a complex and dynamic interplay of epi- extracellular wound matrix facilitates cell adhesion, migration, thelial and mesenchymal cells in concert with tissue-resident and and proliferation. Contemporaneously to the formation of granu- recruited hematopoetic cells to accomplish the sequential phases of lation tissue, at the wound edge, complex epidermal-mesenchymal the repair response: inflammation, tissue formation, and maturation interactions stimulate keratinocyte proliferation and migration to (1–4). The early stage of the repair response is dominated by the restore the epidermal barrier (5). Granulation tissue formation inflammatory phase, which is characterized by local activation of continues until the wound space is refilled and the overlaying the innate immune system, resulting in an immediate influx of epidermis is restored. Upon completion of the epidermal barrier, by guest on September 24, 2021 polymorphonuclear leukocytes (neutrophils) followed by sub- the repair response enters the late stage, which is characterized by sequent invasion of blood monocytes, which differentiate into tissue maturation. During the phase of tissue maturation, granu- tissue macrophages. The mid-stage of the repair response consists lation tissue transforms into scar tissue, characterized by attenu- of the phase of tissue formation, which is characterized by the ated cell proliferation, inflammation, neovascularization, and development of granulation tissue that refills the dermal wound replacement of the provisional matrix by deposition of collagen. space. Granulation tissue formation encompasses the invasion of Numerous studies in the past revealed functional consequences endothelial cells resulting in angiogenesis, the influx of fibro- of the innate immune response of the resident cells as well as the recruited inflammatory cells during skin repair (6). They combat x invading microbes, contribute to debris scavenging, but may also *Department of Dermatology and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne; ‖Medical critically support the repair process by releasing a spectrum of Faculty Carl Gustav Carus, Institute for Immunology, Technische Universita¨t Dres- growth factors. However, due to the release of proinflammatory den, Dresden; †First Medical Department, University of Mainz, Mainz, Germany; ‡Department of Immunology and Molecular Pathology, University College London, and cytotoxic mediators, uncontrolled activity of macrophages London; and{Faculty of Life Sciences, University of Manchester, Manchester, United may also be detrimental to tissue repair. Indeed, imbalanced in- Kingdom flammation characterized by increased numbers of macrophages is Received for publication October 13, 2009. Accepted for publication January 25, a hallmark of an attenuated repair response in human diseases 2010. encompassing diabetes mellitus, vascular disease, and aging (7, 8). This work was supported by the Deutsche Forschungsgemeinschaft (SFB829 to S.A.E. Currently, it is not completely understood how macrophages ex- and T.K. and SFB/TR 52 to A.W.). actly impact the physiology of the repair response and how they Address correspondence and reprint requests to Dr. Sabine A. Eming, Professor of Dermatology, University of Cologne, Jospeh-Stelzmann Strasse 9, 50931 Cologne, may contribute to the pathology of the repair response in diseased Germany. E-mail address: [email protected] conditions. Therefore, a more thorough understanding of macro- The online version of this article contains supplemental material. phage-specific functions during the diverse phases of the repair Abbreviations used in this paper: d, dermis; DT, diphtheria toxin; e, epidermis; Fizz1, response might broaden the view of this cell type in skin physi- found in inflammatory zone 1; g, granulation tissue; he, hyperproliferative epidermis/ ology and pathology. epidermal wound edge; HPF, high-power field; iDTR, inducible diphteria toxin re- ceptor; LysM, lysozyme M; pc, panniculus carnosus; rp, red pulp; sc, s.c. fat muscle Neutrophils and macrophages represent the major fraction of layer; sm, s.c. muscle layer; a-SMA, a smooth muscle actin; st, scar tissue; TbRII, inflammatory cells recruited into the wound site. Within a few TGF-b receptor type II; VEGF-A, vascular endothelial growth factor-A; wp, white hours postinjury, neutrophils transmigrate across the endothelial pulp; Ym1/ECF, eosinophil chemotactic factor. cell wall and begin the debridement of devitalized tissue and in- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 fectious agents. Whereas the presence of neutrophils at the wound www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903356 The Journal of Immunology 3965 site is timely restricted to the early stage of the wound healing phycocyanin-conjugated anti-CD115 (eBioscience, San Diego, CA). Cells response, macrophages persist through all stages of the repair were incubated for 30 min at 4˚C and washed three times thereafter in response. Their number increases during the phase of in- PBS (0.5% BSA and 2 mM EDTA). The cells were analyzed on a FACSCalibur using CellQuest Pro Software (BD Biosciences, Heidel- flammation, peaks during the phase of tissue formation, and berg, Germany). gradually declines during the maturation phase (9). Experiments in the 1970s established the concept that under Wound phase-restricted depletion of macrophages sterile conditions, the influx of macrophages is essential for effi- To characterize macrophage function during distinct phases of repair, we cient healing of incisional skin wounds, whereas the influx of inflicted full-thickness excisional skin wounds on the back of LysMCre/ neutrophils might not be crucial (10, 11). This dogma has been iDTR or LysMCre mice (referred to as control mice). To achieve wound phase-restricted depletion of macrophages, LysMCre/iDTR and control challenged by recent reports, thereby arguing against an essential mice were injected with